Process for preparing bioactive protein-enriched whey products

ABSTRACT

The present invention involves the discovery that various liquid (whey) streams drained or expelled from cheese curd after salt addition in the preparation of cheese contain enriched levels of bioactive proteins such as lactoferrin, lactoperoxidase, immunoglobulins, and growth factors. According to the invention, these proteins may be further enriched through manipulation of the cheese salting process as described herein. The methods of the invention may be used to produce various whey products with enriched levels of all the above bioactive proteins present and, through manipulation of salting conditions, to enrich these proteins selectively.

BACKGROUND OF THE INVENTION

The proteins present in milk, colostrum, whey, and other suchcompositions produced from lactating animals, are of great value. Theyhave unique nutritional and functional properties and are often used asingredients in processed and prepared foods, as well as nutritionalsupplements and even pharmaceutical formulations. These proteins aregenerally categorized into two classes. The first class is aheterogenous mixture called casein and represents approximately 80% ofthe proteins found in milk compositions. The second class is aheterogenous mixture called whey proteins comprising the remaining 20%of the proteins in milk.

Human milk and bovine colostrum contain many bioactive whey proteins,such as lactoferrin (Lf), lactoperoxidase (Lp), immunoglobulins (Ig) andseveral growth factors. Bovine milk also contains these bioactivecomponents, but at much lower levels than human milk and bovinecolostrum.

Large amount of research have been carried out in studying thebiological properties of these bioactive proteins from milk and whey.Harper (2000) has reviewed extensively research on many aspects of thepotential benefits of these proteins. These bioactive proteins have beenshown to possess many biological activities. For example, bioactiveproteins from milk and whey such as immunoglobulins, lactoferrin, andlactoperoxidase, all have various types of anti-microbial activities.Bioactive proteins from milk and whey such as lactoferrin, transforminggrowth factory-β (TGF-β), and immunoglobulins have immune-modulation orimmune enhancing effects. Bioactive proteins such as growth factors inwhey have been shown to stimulate growth of tissue and cultured cells.There is increasing evidence that some bioactive proteins from milk andwhey may have therapeutic value in the treatment of different types ofcancer.

Researchers have employed techniques involving various types ofchromatography to extract or purify the bioactive proteins from milk.For example, Morrison et al. (1963), Johansson (1969), Law and Reiter(1977) and Elliot et al. (1984) describe ways to extract lactoferrin orlactoperoxidase from various milk sources using cation exchangechromatography. Jin et al. (1991) and Francis et al. (1995) describemethods to extract growth factors from milk or whey usingcation-exchange chromatography as the major step.

There are many patents related to extraction, purification, orenrichment of bioactive proteins from milk. For instance, Peyrouset etal. (U.S. Pat. No. 4,436,658) describe a process employing a weaklycation-exchange medium to isolate lactoferrin, lactoperoxidase, andimmunoglobulins from whey. Okonogi et al. (U.S. Pat. No. 4,791,193)describe a process, also using cation-exchange medium, to produce highpurity lactoferrin from skim milk or whey. Uchida et al. (U.S. Pat. No.5,516,675) isolated lactoperoxidase, secretory component and lactoferrinfrom skim milk or whey using a cation-exchange resin. Ballard et al.(U.S. Pat. No. 5,866,418, U.S. Pat. No. 6,447,808) and Read et al. (U.S.Pat. No. 6,183,784) describe various pharmaceutical uses of milkextracts with high levels of growth factors or growth promotingactivities made by cation-exchange chromatography of milk or whey.Kivits et al. (WO 01/25276) describe extracting transforming growthfactor-β (TGF-β), insulin-like growth factor (IGF-1), lactoperoxidaseand immunoglobulins from skim milk or whey using cation-exchangechromatography and then separating them by a hydroxyapatite column.Maubois (WO 03/006500) describe a process to enrich TGF-β, which processinvolves first treating milk serum protein solution at pH4-5.5 and55-68° C. and then microfiltering the treated solution to obtain a TGF-βenriched retentate.

All of the above processes start with milk, whey, a combination orfraction thereof, and employ costly chromatographic or membraneprocesses to concentrate and/or purify bioactive proteins. The high costof isolation and purification of these bioactive proteins hinders theircommercial utilization. As can be seen, a need exists in the art forsimple processes for isolating bioactive proteins from milk components.

SUMMARY OF THE INVENTION

The present invention involves manipulation of the cheese saltingprocess to obtain a product that is enriched in bioactive proteins.According to the invention, the cheese manufacturing process is used asa way to concentrate bioactive proteins from milk, similar to anion-exchange chromatographic process.

In one embodiment of the invention, a method is provided for preparing asalt whey protein product comprising removing fat from salt whey toprovide clarified salt whey and removing salt from the clarified saltwhey to provide the salt whey protein product, wherein the salt wheyprotein product comprises a bioactive protein. In another embodiment ofthe invention, a method is provided for preparing a salt whey proteinproduct comprising (a) recovering a curd product from a solutioncomprising coagulated milk; (b) contacting the curd product with salt toprovide salted curd and at least one liquid stream comprising salt whey,wherein the salt comprises up to 10% w/w of curd weight; (c) recoveringat least one liquid stream comprising salt whey; and (d) concentratingthe salt whey to provide the salt whey protein product, wherein theproduct is enriched for a target bioactive protein. Also provided areproducts prepared by such methods, including a human nutritionalproduct, a personal care product, a health care product, an animalnutritional product, and a biological product.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B are flow diagrams depicting the preparation of bioactiveprotein enriched salt whey products according to methods of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

During the process of cheese manufacturing, casein and fat present inthe milk starting material form the cheese curd. Whey, which containsthe major whey proteins and lactose, is drained from the cheese curd.Salt is then added to the cheese curd to improve the flavor, controlmicrobial metabolism, and expel moisture from the curd. The liquidexpelled from cheese curd after salt addition is referred to as saltwhey. Traditionally, “salt whey” is often either mixed with the bulk ofthe whey to be further processed into whey products, or segregated fromthe bulk of the whey and disposed as waste.

It has been discovered by the present inventors that salt whey isenriched for many bioactive proteins, such as lactoferrin,lactoperoxidase, immunoglobulin, and growth factors, e.g., TGF-β, andIGF-1, as compared to the levels of these bioactive proteins in whey. Itwas also discovered by the present inventors that a salt whey product,such as salt whey product enriched with one or more of theabove-mentioned bioactive proteins can be made without the costly andtime consuming need for complicated fractionation processes. Allprevious methods of purifying and enriching various bioactive proteinsfrom milk or whey involve the use of ion-exchange or microfiltrationfractionation processes. The current invention discloses the use of araw material stream from the cheese manufacturing process, which isenriched in bioactive components. According to the present invention,bioactive protein enriched whey products can be made without using theprocesses of ion-exchange and/or membrane fractionation, resulting inlower product cost and much greater efficiency.

The present inventors also have discovered that during cheesemanufacture, the cheese salting process can be manipulated so as totarget one or more bioactive proteins and provide a selectively enrichedsalt whey protein product.

According to the methods of the invention, salt whey is collected fromany cheese manufacturing process and is retained. The salt whey is firstclarified to remove fat using methods known in the art. The clarified(and optionally Pasteurized) salt whey is then concentrated to removesalt and lactose, traditionally by ultrafiltration (UF). The molecularweight cut-offs (MWCO) of the ultrafiltration membranes used in thisstep are below those of the bioactive proteins intended to recover. Inone embodiment of the invention, MWCO the membranes are 3K to 30K.Diafiltration (DF) can also be used to remove most of the salt. Theresulting protein product can optionally be dried by freeze-drying orspray drying.

Pharmaceutical compositions comprising the bioactive protein enrichedproduct of the invention can be formulated using standard techniques.These typically involve the combination of the product with aphysiologically compatible carrier for administration.

I. Definitions

Unless defined otherwise, all technical and scientific terms used hereinshall have their same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods and materials are described. For purposes of the presentinvention, the following terms are defined below.

As used herein, the term “biologically active protein,” “bioactiveprotein,” “biologically active protein fragment” or “bioactive proteinfragment” is any polypeptide or fragment thereof derived from a saltwhey solution according to the teaching of this invention that hasbiological activity, e.g., enzymatic activity, etc. Thus, the term“bioactive protein” refers to a protein having biological activity thatmay be purified from salt whey according to the teachings and methodsherein. This can include but is not limited to, lactoferrin,lactoperoxidase, immunoglobulins, growth factors and the like. The termalso includes recombinant proteins that are present in milk startingmaterial for any reason.

“Whey” is used herein as a collective term referring to the serum orwatery part of milk that remains after coagulation, which occursprimarily in the production of cheese and cottage cheese. In general,whey is composed of lactose (milk sugar), minerals, vitamins and “wheyproteins.” For example, milk whey protein is prepared by removing fatand casein from milk, and comprises α-lactalbumin, β-lactoglobulin andwhey albumin. β-lactoglobulin (β-lg) constitutes about 50% of the totalwhey proteins.

“Salt whey” and “salt whey solution” refer to a solution containing saltwhey. Cheese production is a biochemical process in which milk isconverted to a solid intermediate product known as curd. After aninitial liquid by-product, referred to as “whey,” “raw whey” or “naturalcheese whey,” is drained from the curd, a salt is added to remove anadditional solution from the curd. The additional solution is referredto as “salt whey.” Thus, the term “salt whey” refers to any liquid thatis drained and/or expelled from cheese curd after the addition of saltin the preparation of cheese or other such milk-related products thatare prepared in a similar manner to cheese.

“Coagulation,” as used herein, refers to the conversion of any solubleprotein to an insoluble form of the protein.

As used herein, the term “enriched” refers to a composition or fractionwherein an object species has been partially purified such that, on aweight basis, the concentration of the object species is substantiallyhigher than the naturally occurring level of the species in a finishedproduct without fractionation. For example, when processing normalcheese whey into whey protein concentrate (WPC) without anyfractionation, 0.1 to 1.0% of the protein in the final WPC product islactoferrin. Therefore, the “naturally-occurring” level of lactoferrinin WPC is considered to be 0.1 to 1.0% of the protein. The term“naturally-occurring” as used herein as applied to an object refers tothe fact that object can be found in nature. For example, a polypeptideor polynucleotide sequence that is present in an organism (includingviruses) that can be isolated from a source in nature and which has notbeen intentionally modified by man in a laboratory, isnaturally-occurring.

For example, in one embodiment of the present invention, a salt wheyprotein product is produced that is enriched for lactoferrin, e.g., theproduct comprises about 3 to about 20 times the amount of lactoferrinthat is typically present in whey or a whey protein product. In anotherembodiment of the present invention, a salt whey protein product isproduced that is enriched for lactoperoxidase, e.g., the productcomprises about 2-5 times the amount of lactoperoxidase that istypically found in whey or a whey protein product. In another embodimentof the present invention, a salt whey protein product is produced thatis enriched for insulin-like growth factor 1 (IGF-1), e.g., the productcomprises about 2-10 times the amount of IGF-1 that is typically foundin whey or a whey protein product. In another embodiment of the presentinvention, a salt whey protein product is produced that is enriched fortransforming growth factor β1 (TGF-β1), e.g., the product comprises upto 40 times the amount of TGF-β1 that is typically found in whey or awhey protein product. In another embodiment of the present invention, asalt whey protein product is produced that is enriched for transforminggrowth factor β2 (TGF-β2), e.g., the product comprises up to 10 timesthe amount of TGF-β2 that is typically found in whey or a whey proteinproduct. In another embodiment of the present invention, a salt wheyprotein product is produced that is enriched for immunoglobin G (IgG),e.g., the product comprises up to 2 times the amount of IgG that istypically found in whey or a whey protein product.

As used herein, the term “milk” shall include any composition capable ofbeing excreted from the mammary gland of a lactating animal. Forexample, milk from a cow, sheep, goat, buffalo, deer, camel, horse,llama, yak, or any mixture thereof.

As used herein, “substantially pure” means that an object species is thepredominant species present (i.e. on a molar basis, it is more abundantthan any other individual species in the composition), and preferably assubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50% (on a weight basis) of allmacromolecular species present. Generally a substantially purecomposition will comprise more than about 80-90% of all macromolecularspecies present in the composition. Most preferably the object speciesis purified to essential homogeneity (contaminant species cannot bedetected in the composition by conventional detection methods) whereinthe composition consists essentially of a single macromolecular species.

II. Methods of the Present Invention

The process of cheese manufacture is well known in the art and isdescribed more fully in the references incorporated herein.

Briefly, the starting material for cheese production is milk orconcentrated milk suitable for cheese manufacture. After the milk isclotted (coagulated) by the action of clotting enzymes known andstandardly used in the art, most of the whey is drained, and a givenlevel of salt is added to the remaining curd.

The level of salt to be added can be determined based on the requirementfor cheese production, and could range from about 1% to about 10%. Inone embodiment, salt is added in a range of about 1% to about 3% of thecheese weight. To achieve the desired enrichment levels of the bioactiveproteins, salt is added after most of the whey (>90%) has been removedfrom the curd. Although sodium chloride is the most commonly used saltin cheese making, many other salts can be employed in the method of thepresent invention. Examples of the other salts include, but are notlimited to, potassium chloride, ammonium chloride, calcium chloride,sodium citrate, etc.

According to the invention, after salt addition, the salt whey isdrained naturally, e.g., through perforated belts or screen, andcollected. Various liquid streams can be collected after the saltaddition step, and all of which are referred to as salt whey streams,which may be used according to the invention. Additional liquid saltwhey may be pressed out by mechanical means from the curd and collected.The salt whey streams may be pooled and processed together to produce asingle product with enriched levels of various bioactive proteins. Inanother embodiment of the invention, at least two salt whey streams canbe collected and processed separately into salt whey products havingdifferent levels of enriched bioactive proteins of interest (FIG. 1B).For example, salt whey that is naturally drained and that ismechanically pressed out can be processed into two different products.Optionally, salting can be achieved in two steps: a lower level of saltaddition and collection of the liquid followed by a higher level of saltaddition and collection of liquid. The two liquid streams collected atdifferent salt levels can be processed separately to produce productswith selective enrichment of various bioactive proteins.

According to the present invention, the processing of salt whey streamsinvolves filtration, such as nanofiltration or ultrafiltration withproper membrane pore sizes, to remove the salts (and lactose in case ofultrafiltration) into permeate. Retentate of this filtration process canbe dried by spray drying or freeze-drying into a powder. The conditionsfor filtration and drying processes need to be carefully controlled tofully recover the bioactive proteins in the final products.

When processed according to present invention using various salt wheystreams, the finished products (such as 60-80% whey proteinconcentrates) can have about 3 to about 20 times enrichment inlactoferrin, about 2 to about 5 times enrichment in lactoperoxidase,about 2 to about 10 times enrichment in IGF-1, up to 40 times enrichmentof TGF-β1, up to 10 times enrichment in TGF-β2, and/or up to 2 timesenrichment in IgG as compared to the corresponding products made fromwhey.

Currently, bioactive proteins such as those described herein areobtained from milk using a variety of costly processing techniques. Mostof these techniques are based upon chromatographic or membranefractionation techniques. The following is a list of separationtechniques currently used in the art of protein purification from milkproducts and may be used to recover the salt whey bioactive proteinenriched product by the methods of the present invention.

1. Ultrafiltration (UF) designates a membrane separation process drivenby a pressure gradient in which components of a liquid are fractionatedas a function of their solvated size and structure. In UF, the membranepore size is large enough to allow some components of a liquid to passthrough with the water. For example, when concentrating whey proteinusing a 10,000 MW cutoff polysulfone membranes, most of the lactose andminerals will pass through the membrane to the permeate, some of thesmaller polypeptides will also pass through the membrane, whereas almost100% of protein and fat will remain in the retentate.

2. Microfiltration (MF) designates a membrane separation process similarto UF but with larger membrane pore size allowing particles in the rangeof 0.02 to 2 micrometers to pass. The pressure used is generally lowerthan that of UF process. MF is used in the dairy industry for makinglow-heat sterile milk as proteins may pass through the membrane but notbacteria.

3. Chromatography. Fractionation may also be accomplished using avariety of chromatographic techniques. The most commonly usedchromatographic process for industrial protein fractionation ision-exchange chromatography. It relies on an inert matrix with chargedfunctional groups to adsorb oppositely charged protein molecules. The pHof the protein feed solution is selected so that the proteins to beadsorbed will be charged oppositely to that of the ion-exchange resin.Following adsorption and separation of the non-adsorbed protein andother components in the original protein feed solution, the adsorbedproteins are eluted with salt solution or by changing the pH so theprotein charge properties change.

The following examples are intended to illustrate but not limit theinvention.

EXAMPLES

Materials and Methods

HPLC and immunochemical methods were used for the analysis of all theproteins and protein fractions isolated from whey.

α-lactalbumin (α-lα) and β-lactoglobulin (β-lg) in all the whey proteinproducts were measured by HPLC with a size-exclusion column (Shodex)using a mobile phase of 6 M urea, 0.1 M phosphate buffer, and 0.1 Msodium sulfate, pH 6.0. The samples are dissolved in 0.1 M phosphatebuffer, pH 6.0, containing 6 M urea, 0.1 M sodium sulfate, and 0.2%dithiothreitol (DTT) and heated at 80C for 5 minutes to fully denaturethe proteins.

Immunoglobulin G (IgG) and bovine serum albumin (BSA) were analyzed bythe same HPLC column, but under native conditions using a mobile phaseof 0.1 M phosphate buffer and 0.1 M sodium sulfate, pH 6.0. IgG was alsoanalyzed using radial immuno diffusion (RID) test from Triple-J Farm(Bellingham, Wash.)

Lactoferrin (Lf) and lactoperoxidase (Lp): HPLC with an ion-exchangecolumn (Mono-S from Pharmacia) was used to measure Lf and Lp in thesamples. Samples were dissolved in 0.1 M phosphate buffer, pH 7.0 andlactoferrin and lactoperoxidase were eluted by the phosphate buffer witha 0.05 M to 1.80 M NaCl gradient.

Growth factors IGF-1, TGF-beta1 and TGF-beta2 were measured by ELISAmethods. IGF-1 was measured using the test kit and its protocol from R&DSystems (Minneapolis, Minn.). TGF-beta1 and TGF-beta2 were both measuredusing the test kits and protocols of Emax® ImmunoAssay Systems for TGFβ1and TGFβ2 from Promega Corporation (Madison, Wis.).

The control whey protein concentrate (WPC) used in the comparison iscommercially available (Proliant 8000 Whey Protein Concentrate 80%,Proliant, Inc., Ames Iowa).

Referring to FIGS. 1A and B, in the process of cheese manufacture, thestarting material is milk. The milk can be Pasteurized, and a starterculture can be added. After the milk has coagulated, most of the whey isdrained, and an optional rinse with water is carried out (optional rinse(I) in FIGS. 1A and B). A salt solution can be sprayed onto the curd(salt solution I in FIGS. 1A and B). The concentration of salt at thisstep can be from 0.5% to 10%, e.g., 0.5% to 5%, of the curd weight andis determined based on the type of bioactive proteins that are to berecovered and/or enriched, as well as the requirement of cheese flavordevelopment. After the salt addition, the liquid drained naturally(salting/drain/II, in FIG. 1A) and the liquid pressed out in furthercheese process (pressing curd (11) in FIG. 1A) can be pooled andprocessed together to produce a single product with enriched level ofvarious bioactive proteins. Alternatively, the two liquid stream can becollected and processed separately into two whey products with differentlevels of enrichment of various bioactive proteins.

Optionally, cheese process and salting processes can be modified toobtain streams of salt whey with selective enrichment of variousbioactive proteins. Examples of such modification includes changing thepH at which most of the whey is drained to achieve selective enrichmentof desired bioactive proteins, and a two step salting process with alower level of salt addition and collection of the liquid followed by ahigher level of salt addition. The two liquid streams collected atdifferent salt levels can be processed separately to produce productswith selective enrichment of various bioactive proteins. For example, alower level of salt addition and collection of the liquid (salt whey 1in FIG. 1B) followed by a higher level of salt addition (salt whey 2 inFIG. 1). Salt whey 1 and salt whey 2 in FIG. 1B are then processedseparately to produce products with selective enrichment of variousbioactive proteins.

The collected salt whey streams are clarified to remove fat. Theclarification can be achieved through centrifugal separation ormicrofiltration. The clarified (and optionally Pasteurized) streams areconcentrated through ultrafiltration (UF) to remove salt and lactose.The molecular weight cut-offs (MWCO) of the membranes are below those ofthe bioactive proteins of interest, for example, 3K to 30K.Diafiltration (DF) is used to remove most of the salt from the proteinproduct. The product can then be dried by freeze-drying or spray drying.

As discussed herein, the starting material for cheese manufacture ismilk, which in one embodiment of the present invention is Pasteurized.In a typical cheese production process starter culture is added to themilk.

Example 1

Salt whey pressed out in Cheddar cheese process was collected from thetower. The salt whey was centrifuged at 7000×g for 15 minutes at 10° C.to remove fat. After adjusted to pH 6.2 with 6 N NaOH, the separatedsalt whey was concentrated by ultrafiltration and diafiltration using abench UF unit with a 10K MWCO spiral-wound membrane cartridge. The finalconcentrate was freeze-dried into a protein powder (salt whey product A;Table 2).

Example 2

The salt whey streams naturally drained from curd and pressed out inlater cheese processing were pooled together and separated by a pilotplant separator (Westfalia SA-7). The separated salt whey wasconcentrated by ultrafiltration and diafiltration using a pilot plant UFunit with two 10K MWCO spiral-wound membranes. The final concentrate wasspray-dried into a protein powder (salt whey product B; Table 2).

Example 3

The salt whey pressed out in cheddar cheese process was collected fromthe tower. It was separated by a pilot plant separator (Westfalia SA-7).The separated salt whey was concentrated by ultrafiltration anddiafiltration using a pilot plant UF unit with two 10K MWCO spiral-woundmembranes. The final concentrate was spray-dried into a protein powder(salt whey product C; Table 2).

Example 4

The salt whey naturally drained was collected from cheese belt. It wasseparated by a pilot plant separator (Westfalia SA-7). The separatedsalt whey was concentrated by ultrafiltration and diafiltration using apilot plant UF unit with two 10K MWCO spiral-wound membranes. The finalconcentrate was spray-dried into a protein powder (salt whey product D;Table 2).

Example 5

The salt whey naturally drained and pressed out in later cheeseprocessing was pooled together. It was separated by a commercial scaleseparator. A portion of the separated salt whey was concentrated byultrafiltration and diafiltration using a pilot plant UF unit with two10K MWCO spiral-wound membranes. The final concentrate was spray-driedinto a protein powder (salt whey product E; Table 2). TABLE 2Compositions of bioactive protein enriched WPC samples IGF1 TGF-β1TGF-β2 Salt Whey Lf % prot Lp % prot IgG ng/g ng/g ng/g Product Protein% Ash % % protein % protein % protein protein protein protein A 63.658.03 32.05 0.84 4.45 284 6045 881 B 67.19 1.81 17.92 0.64 5.34 532 46393073 C 62.31 3.00 13.63 0.74 4.94 794 4872 2648 D 62.26 1.48 20.03 0.055.27 223 7295 2435 E 73.16 1.73 25.35 0.65 5.95 202 8188 2008 Control76-80 2.5-5 <0.5 <0.1 3-5 177 150 160 WPC

Example 6

In the cheese manufacturing process, after the milk is coagulated andthe whey is drained, water of up to 100% of the curd weight is sprayedonto the curd. The liquid is drained and combined with regular whey.Salt is added at 1% to 10% of curd weight after most of the liquid isdrained. The salt whey that is naturally drained and pressed out inlater cheese processing is then pooled together, separated by aseparator, and concentrated by ultrafiltration and diafiltration with10K MWCO spiral-wound membranes. The final concentrate is spray-driedinto a protein powder. Such a salt whey product is expected to have upto 3 times enrichment of all the bioactive protein listed in Table 2compared to the samples A-E produced in Examples 1-5.

Example 7

In the cheese manufacturing process, after the milk is coagulated andthe whey is drained, a salt solution of up to 100% of the curd weight issprayed onto the curd. The concentration of the salt solution is suchthat it will provide an equivalent of up to 2% of the curd weight ofsalt (NaCl). The liquid drained after this first salt solution rinse iscollected as Salt Whey A. Once most of salt whey A is drained, apowdered salt is added to the curd at up to 10% of curd weight. Theliquid drained and pressed out after the second salt addition is pooledtogether and collected as Salt Whey B. Both Salt Whey A and Salt Whey Bare separated by a separator and concentrated by ultrafiltration anddiafiltration with 10K MWCO spiral-wound membranes. The two finalconcentrates can be spray-dried into two protein powders, SWP A and SWPB, respectively. Compared to the bioactive protein compositions ofsamples made in example 1-5 (Table 2), SWP A is expected to haveincreased levels of IgG and Lp, but a reduced level of Lf, whereas SWP Bis expected to have decreased levels of IgG and Lp, but a much increasedlevel of Lf. Depending on the amount of salt used in the first addition,either to SWP A or SWP B, can be selectively enriched for growth factorsIGF-1, TGFβ1 and TGFβ2 as compared to samples A-E in example 1-5.

REFERENCES

Morrison, M., and Hultquist, D. E. 1963. Lactoperoxidase. II. Isolation.J. Biol. Chem. 238(8), 2847-2847.

Harper, J. 2000. Biological Properties of Whey Components, A Review.American Dairy Products Institute.

Johansson, B. G. 1969. Isolation of crystalline lactoferrin from humanmilk. Acta Chem. Scan. 23, 683.

Law B. A. and B. Reiter 1977. The isolation and bacteriostaticproperties of lactoferrin from bovine milk whey. J. Dairy Res. 44, 595.

Elliot, J. I., Senft, B., Erhardt, G., and Fraser, D. 1984. Isolation oflactoferrin and its concentration in sows' colostrum and milk during a21 -day lactation. J. Animal Sci. 59(4), 1080-1084.

Jin, Y., Cox, D. A., Knecht, R., Raschdorf, F., and Cerletti, N. 1991.Separation, purification and sequence identification of TGF-β1 andTGF-β2 from bovine milk. J. Protein Chem. 10(5), 565-575.

Francis, G. L., Regester, G. O., Webb, H. A., and Ballard, F. J. 1995.Extraction from cheese whey by cation-exchange chromatography of factorsthat stimulate the growth of mammalian cells. J. dairy Sci. 78,1209-1218.

All publications, patents and patent documents are incorporated byreference herein, as though individually incorporated by reference. Theinvention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

1. A method for preparing a salt whey protein product comprising:removing fat from salt whey to provide clarified salt whey; and removingsalt from the clarified salt whey to provide the salt whey proteinproduct, wherein the salt whey protein product comprises a bioactiveprotein.
 2. The method of claim 1 wherein the bioactive proteincomprises lactoferrin, lactoperoxidase, albumin, a growth factor or animmunoglobulin.
 3. The method of claim 2 wherein the protein product isenriched for lactoferrin.
 4. The method of claim 2, wherein the proteinproduct is enriched for lactoperoxidase.
 5. The method of claim 2,wherein the growth factor is insulin-like growth factor 1 (IGF-1),transforming growth factor β1 (TGF-β1) or transforming growth factor β2(TGF-β2).
 6. The method of claim 2, wherein the protein product isenriched for insulin-like growth factor 1 (IGF-1).
 7. The method ofclaim 2 wherein the protein product is enriched for transforming growthfactor β1 (TGF-β1).
 8. The method of claim 2 wherein the protein productis enriched for transforming growth factor β2 (TGF-β2).
 9. The method ofclaim 2 wherein the protein product is enriched for immunoglobin G(IgG).
 10. The method of claim 1 further comprising drying the salt wheyprotein product.
 11. The method of claim 1 wherein the protein productis dried by spray drying or freeze drying.
 12. The method of claim 1,further comprising concentrating the salt whey protein product.
 13. Themethod of claim 12, wherein the product is concentrated byultrafiltration (UF), diafiltration, or a combination thereof.
 14. Thesalt whey protein product prepared by the method of claim
 1. 15. Amethod for preparing a salt whey protein product comprising: a.recovering a curd product from a solution comprising coagulated milk; b.contacting the curd product with salt to provide salted curd and atleast one liquid stream comprising salt whey, wherein the salt comprisesup to 10% w/w of curd weight; c. recovering at least one liquid streamcomprising salt whey; d. concentrating the salt whey to provide the saltwhey protein product, wherein the product is enriched for a targetbioactive protein.
 16. The method of claim 15, wherein the targetbioactive protein is concentrated from the curd product followingcontact with the salt, wherein the salt is present at up to 2% w/w ofcurd weight.
 17. The method of claim 15, further comprising contactingthe salted curd with additional salt, and recovering a second liquidstream comprising salt whey.
 18. The method of claim 15, furthercomprising modifying the pH of the coagulated milk solution.
 19. Themethod of claim 15, further comprising removing fat from the recoveredliquid stream comprising salt whey.
 20. The method of claim 15, whereinthe target bioactive protein comprises lactoferrin, lactoperoxidase,albumin, a growth factor or an immunoglobulin.
 21. The salt whey proteinproduct produced by the method of claim
 15. 22. A human nutritionalproduct comprising: a salt whey protein product manufactured accordingto the method of claim 1 or claim 15; wherein the nutritional product isselected from the group consisting of infant formula, adult nutritionalproducts, medical nutritional formula, nutritional supplement products,and nutritional foods.
 23. A personal care product comprising: a saltwhey protein product manufactured according to the method of claim 1 orclaim 15, wherein the care product is used for wound healing, cosmetics,dental health, and other topical applications.
 24. A health care productcomprising: a salt whey protein product manufactured according to themethod of claim 1 or claim 15, wherein the care product is used inapplications such as reduction of risk factors for cancer,cardiovascular diseases, ulcer, and reduction of side effects duringtreatment of cancer or chemotherapy.
 25. An animal nutritional productcomprising: a salt whey protein product manufactured according to themethod of claim 1 or claim 15, wherein the nutritional product is usedto improve feed utilization, increase carcass yield, and reduce the useof antibiotics.
 26. A biological product comprising: a salt whey proteinproduct manufactured according to the method of claim 1 or claim 15, ora modified version of it; wherein the biological product is used inbiotechnology industry for fermentation or cell culture media.